The electrical behavior of cells and cell membranes is of profound importance in basic research as well as in modem drug development. As described in the above-reference parent applications, a specific area of interest in this field is in the study of ion channels and transporters. Ion channels are protein-based pores found in the cell membrane that are responsible for maintaining the electro-chemical gradients between the extra cellular environment and the cell cytoplasm.
Quite often these membrane channels are selectively permeable to a particular type of ion, e.g. potassium or sodium. The channel is generally comprised of two parts; the pore itself, and a switch mechanism that regulates (gates) the conductance of the pore. Examples of regulation mechanisms include changes in transmembrane voltage or the activation or deactivation of a membrane receptor via a chemical ligand. Ion channels are passive elements in that once opened, ions flow in the direction of existing chemical gradients. Ion transporters are similar in that they are involved in the transport of ions across the cell membrane, however they differ from ion channels in that energy is required for their function and they tend to actively pump against established electrochemical gradients.
An interesting and technically challenging aspect of ion channels involves their rapid and quite diverse signaling kinetics. Many ion channels can be activated and then de-activated in a few milliseconds. This requires that the instrumentation used in their analysis have the ability to following these changes with a fairly high temporal bandwidth, on the order of 10 kHz.
For an instrument to resolve these kinds of changes it is not only necessary that the recording apparatus have the required temporal bandwidth, but in addition the method of stimulating the ion channel event must also be fast. The electrical recording aspect of this problem is involved but readily achievable since high-bandwidth operational amplifiers are readily available.
The issue of achieving a rapid stimulus deserves additional explanation. As previously mentioned, some ion channels are activated by voltage. In these cases the same electronics used to record ion channel currents can also be used to control the voltage stimulus. This type of measurement is common in the industry and is referred to as a voltage clamp. In this case the time bandwidth of the stimulus, an electrical signal, is inherently fast enough so as not to degrade the kinetics of the voltage-gated ion channel signals.
Another class of ion channel events relies on chemical or “ligand” gating. These kinetic channel events are activated by specific chemical messengers such as the release of intracellular calcium, adenosine 3′,5′—monophosphate (cyclic AMP or cAMP) or acetylcholine (ACh). It is beyond the scope of this application to discuss all of the potential signaling chemicals that are of biological or therapeutic interest and the above serve only as examples. It should be mentioned, however, that in some cases the chemical activation of an ion channel is extra-cellular in its initiation, and in other cases it is intra-cellular. This implies that not only is it important that the compound can be released on the time scale of tens of milliseconds, but in some cases it is desirable to have it introduced within the membrane of a living cell.
One technique for accomplishing rapid stimulation of ligand-gated channels utilizes photo-activatable or “caged” compounds. This term refers to chemicals which are chemically altered such that the active nature of the compound is suppressed (“caged”) until photo-activated, usually by a short pulse of ultra-violet (UV) light of wavelength in the range of 240 and 400 nm.
The photolysis of such compounds is very fast and thereby can rapidly (in some cases in microseconds) release the active species of the compound. The underlying chemistry for making various common biological chemicals photoa-activatable is well-developed and the “caged” version of many compounds are commercially available for purchase through companies such as Molecular Probes of Eugene, Oreg.. In addition, when intracellular application is required, the caged version can often be made cell permeable such that it can be loaded into the cytoplasm of the cell for rapid intra-cellular activation at a later time
The technique of using pulsed UV illumination of a biological sample to rapidly release chemicals is fairly common in the fields of rapid cellular imaging and single-well patch-clamp electrophysiology. Parpura describes a system that utilizes a micro-manipulated optical fiber to deliver UV energy for flash photolysis in support of microscopic imaging studies. U.S. Pat. No. 5,936,728, describes a flash photolysis system for use in a scanning microscope allowing for automatic alignment of the ultraviolet directed beam and the detected image point in time. To date, there has been no utilization of this technique in the field of high-throughput (i.e., non-patch clamp) electrophysiology as described in the parent to this application.